(a) Field of the Invention
The present invention relates to a biosensor specifically detecting protein kinase A (PKA), and a diagnosis kit comprising the biosensor. In particular, the present invention relates to a biosensor for detecting PKA comprising a cantilever, and a peptide derived from the protein kinase inhibitor (PKI) immobilized on the surface of the cantilever; and a kit for diagnosing diseases manifested by the increase of the activity of PKA, comprising a sample, the biosensor, and a quantification method.
(b) Description of the Related Art
Protein kinases play important roles in a variety of intracellular signaling pathways by catalyzing a reaction of a gamma-phosphoryl transfer from ATP to a hydroxyl group at serine, threonine or tyrosine residue of substrate proteins (H. C. Clevers, M. A. Oosterwegel, K. Georgopoulos, Immunol, Today 14 (1993) 592-597). The increase in the protein kinase activity is generally found in a variety of diseases, such as cancers, cardiovascular syndromes, immunological diseases, and hormonal disorders. Therefore, the protein kinases have been considered to be involved in the diseases mentioned above, and there have been extensive works to develop effective inhibitors specifically against thereto. In particular, protein kinase A (PKA) serves as a prototype for protein kinases since it is one of the best characterized members among the large family of protein kinases. In the absence of cAMP, PKA exists predominantly as an inactive tetrameric holoenzyme composed of two regulatory and two catalytic subunits. When the intracellular concentration of cAMP increases, cAMP binds to the regulatory subunits of the inactive PKA holoenzyme, leading to the decrease in the binding affinity between the regulatory and catalytic subunits. This results in the dissociation of two active catalytic subunits from the regulatory subunits to perform various phosphorylation reactions. In addition, the catalytic subunit of PKA can be potently inhibited by the physiological heat-stable inhibitor protein PKI, which may provide a second level of regulation. Based on the observation that certain cancer cells excrete the catalytic subunit of PKA out of cell, it was reported that the extracellular PKA catalytic subunit is correlated with cancers (Yee Sook Cho et al. PNAS 97(2) (2000) 835-840). Since the amount of such extracellular PKA catalytic subunit excreted from the cancel cell is minute, a highly selective detection tool is required for the quantitative and specific detection of the protein.
Various methods have been developed for measuring protein kinase activity. Such methods include a conventional radioactivity-based method that requires physical separation of substrate from its phosphorylated product, an enzyme linked immunosorbent assay (ELISA) that employs phospho-specific antibodies to capture product, and more recently developed fluorescence-based homogeneous nonradioactive assays based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer. Recent technological advances have aided development of numerous methodologies of monitoring interactions between peptides and proteins. In particular, the use of fluorescence polarization-based solution phase measurements and capillary electrophoresis (CE) has been well-established in studies for the peptide-protein binding. However, such direct method of quantitative analysis of an activated kinase is less sensitive than the methods based on signal amplifications by ELISA or an enzyme activity assay. Surface plasmon resonance (SPR) technology has also been recognized as an effective tool for biomolecular interaction analyses, despite the operation complexity. Accordingly, it is needed to develop a highly sensitive and convenient technique of detecting a peptide-protein binding.